Identification and relative contributions of human cytochrome P450 isoforms involved in the metabolism of glibenclamide and lansoprazole: evaluation of an approach based on the in vitro substrate disappearance rate

1. The identification and relative contributions of human cytochrome P450 (CYP) enzymes involved in the metabolism of glibenclamide and lansoprazole in human liver microsomes were investigated using an approach based on the in vitro disappearance rate of unchanged drug. 2. Recombinant CYP2C19 and CYP3A4 catalysed a significant disappearance of both drugs. When the contribution of CYPs to the intrinsic clearance (CL(int)) of drugs in pooled human microsomes was estimated by relative activity factors, contributions of CYP2C19 and CYP3A4 were determined to be 4.6 and 96.4% for glibenclamide, and 75.1 and 35.6% for lansoprazole, respectively. 3. CL(int) of glibenclamide correlated very well with CYP3A4 marker activity, whereas the CL(int) of lansoprazole significantly correlated with CYP2C19 and CYP3A4 marker activities in human liver microsomes from 12 separate individuals. Effects of CYP-specific inhibitors and anti-CYP3A serum on the CL(int) of drugs in pooled human liver microsomes reflected the relative contributions of CYP2C19 and CYP3A4. 4. The results suggest that glibenclamide is mainly metabolized by CYP3A4, whereas lansoprazole is metabolized by both CYP2C19 and CYP3A4 in human liver microsomes. This approach, based on the in vitro drug disappearance rate, is useful for estimating CYP identification and their contribution to drug discovery.     

Approach to predict the contribution of cytochrome P450 enzymes to drug metabolism in the early drug-discovery stage: The effect of the expression of cytochrome b5 with recombinant P450 enzymes

In order to evaluate the potential adverse effects due to genetic polymorphism and/or inter-individual variation, it is necessary to calculate the cytochrome P450 (CYP) contribution to the metabolism of new drugs. In the current study, the in vitro intrinsic clearance (CL_int) values of marker substrates and drugs were determined by measuring metabolite formation and substrate depletion, respectively. Recombinant CYP microsomes expressing CYP2C9, CYP2C19 and CYP3A4 with co-expressed cytochrome b₅ were used, but those expressing CYP1A2 and CYP2D6 did not have co-expressed cytochrome b₅. The following prediction methods were compared to determine the CL_int value using data from recombinant CYP enzymes: (1) relative CYP enzyme content in human liver microsomes; (2) relative activity factor (RAF) estimated from the V_max value; and (3) RAF estimated from the CL_int value. Estimating RAF from CL_int proved the most accurate prediction method among the three tested, and differences in the CYP3A4 marker reactions did not affect its accuracy. The substrate depletion method will be useful in the early drug-discovery stage when the main metabolite and/or metabolic pathway has not been identified. In addition, recombinant CYP microsomes co-expressed with cytochrome b₅ might be suitable for the prediction of the CL_int value.     

Assessment of multiple cytochrome P450 activities in metabolically inactivated human liver microsomes and roles of P450 2C isoforms in reaction phenotyping studies

The fraction of substrate metabolized (f_m) can be used to estimate drug interactions and can be determined by comparison of the intrinsic clearances (CL_int) of victim drugs obtained from inhibited and uninhibited hepatic enzymes_. Commercially available human liver microsomes were recently developed in which one cytochrome P450 (P450) isoform is selectively inactivated. These inactivated liver microsomes were used to evaluate the roles of P450 2C isoforms in the depletion and oxidation of probe substrates. Determination of CL_int with sets of control and P450 2C9‐inactivated liver microsomes yielded f_{m,P450 2C9} values of 0.69–1.0 for celecoxib, diclofenac and warfarin. Apparent minor contributions of P450 1A2/2C8/3A4 were seen in depletion assays, yielding ~1 for the sum of the f_m values. Selectively inactivated liver microsomes were thereby shown to be potentially useful for determining the in vitro f_m values for major P450 2C9 contributions to substrate oxidations. Metabolite formations from diclofenac and warfarin were suppressed by 62–84% by the replacement of control liver microsomes with P450 2C9‐inactivated liver microsomes. R‐, S‐ and racemic omeprazole and troglitazone oxidation activities by liver microsomes at multiple substrate concentrations were suppressed by 26–36% and 22–50%, respectively, when P450 2C19‐ and 2C8‐inactivated liver microsomes were used in place of control liver microsomes. This study provides important information to help elucidate the different roles of P450 isoforms in metabolite formation at different substrate concentrations. The data obtained allow the fractions metabolized to be calculated for victim drugs.     

Use of a cocktail of probe substrates for drug-metabolizing enzymes for the assessment of the metabolic capacity of hepatocyte preparations

1.?A cocktail of the following probe substrates for human drug-metabolizing enzymes was used to characterize hepatocyte preparations: phenacetin (for CYP1A2), diclofenac (CYP2C9), diazepam (CYP2C19), bufuralol (CYP2D6), midazolam (CYP3A4/5) and 7-hydroxycoumarin (for glucuronidation and sulphation).

2.?The cocktail was incubated with cryopreserved human, dog or minipig hepatocytes or with freshly prepared rat hepatocytes. Sample analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in an Open Access environment that allowed less experienced MS operators to login, submit and analyse sample sets using predefined settings without the immediate attendance of an experienced analyst. Intrinsic clearances (CL_int) were calculated from the disappearance of the compounds from the incubations.

3.?Initially, the cocktail used for human, rat and dog hepatocyte incubations contained 7-ethoxycoumarin instead of 7-hydroxycoumarin. However, 7-ethoxycoumarin had an inhibitory effect on the metabolism of phenacetin.

4.?The highest CL_int estimated with human and dog hepatocytes was observed for 7-hydroxycoumarin. For rat and minipig hepatocytes, the highest CL_int was observed for bufuralol. In incubations with dog and minipig hepatocytes, the lowest CL_int was seen with diclofenac, whereas for human and rat hepatocytes, the lowest value was observed with diazepam and phenacetin, respectively.

5.?When the cocktail was incubated together with human hepatocytes and 1?μM ketoconazole, the CL_int of midazolam was decreased to about 7.5% of the control value, whereas the metabolism of the other cocktail compounds was virtually unaffected by this CYP3A inhibitor.

6.?It is suggested that a cocktail of specific human probe substrates for drug-metabolizing enzymes can be used routinely for the determination of the metabolic capacity of hepatocyte preparations in order to ensure the quality and reproducibility of experiments. Moreover, a cocktail of specific probe substrates can also be a useful tool for studies on enzyme inhibition.     

Species differences in hepatic and intestinal metabolic activities for 43 human cytochrome P450 substrates between humans and rats or dogs

Abstract

1. Prediction of human pharmacokinetics might be made more precise by using species with similar metabolic activities to humans. We had previously reported the species differences in intestinal and hepatic metabolic activities of 43 cytochrome P450 (CYP) substrates between cynomolgus monkeys and humans. However, the species differences between humans and rats or dogs had not yet been determined using comparable data sets with sufficient number of compounds.

2. Here, we investigated metabolic stabilities in intestinal and liver microsomes obtained from rats, dogs and humans using 43 substrates of human CYP1A2, CYP2J2, CYP2C, CYP2D6 and CYP3A.

3. Hepatic intrinsic clearance (CL_int) values for most compounds in dogs were comparable to those in humans (within 10-fold), whereas in rats, those for the human CYP2D6 substrates were much higher and showed low correlation with humans. In dog intestine, as with human intestine, CL_int values for almost all human CYP1A2, CYP2C, CYP2D6 substrates were not determined because they were very low. Intestinal CL_int values for human CYP3A substrates in rats and dogs appeared to be lower for most of the compounds and showed moderate correlation with those in humans.

4. In conclusion, dogs showed the most similar metabolic activity to humans.     

Comparison of predicted intrinsic hepatic clearance of 30 pharmaceuticals in canine and feline liver microsomes

1.?Known cytochrome P450 (CYP) substrates in humans are used in veterinary medicine, with limited knowledge of the similarity or variation in CYP metabolism. Comparison of canine and feline CYP metabolism via liver microsomes report that human CYP probes and inhibitors demonstrate differing rates of intrinsic clearance (CL_int).

2.?The purpose of this study was to utilize a high-throughput liver microsome substrate depletion assay, combined with microsomal and plasma protein binding to compare the predicted hepatic clearance (CL_hep) of thirty therapeutic agents used off-label in canines and felines, using both the well-stirred and parallel tube models.

3.?In canine liver microsomes, 3/30 substrates did not have quantifiable CL_int, while midazolam and amitriptyline CL_int was too rapid for accurate determination. A CL_hep was calculated for 29/30 substrates in feline microsomes. Overall, canine CL_hep was faster compared to the feline, with fold differences ranging from 2–20-fold.

4.?A comparison between the well-stirred and parallel tube model indicates that the parallel tube model reports a slighter higher CL_hep in both species.

5.?The differences in CYP metabolism between canine and feline highlight the need for additional research into CYP expression and specificity.     

Role of human cytochrome P450 3A4 in the metabolism of DA–8159, a new erectogenic

1.?The purpose of this paper was to characterize cytochrome P450 (CYP) enzymes involved in N-dealkylation of a new oral erectogenic, DA-8159 to DA-8164, a major circulating active metabolite, in human liver microsomes and to investigate the inhibitory potential of DA-8159 on CYP enzymes.

2.?CYP3A4 was identified as the major enzyme responsible for DA-8159 N-dealkylation to DA-8164 based on correlation analysis and specific CYP inhibitor and antibody-mediated inhibition study in human liver microsomes, and DA-8159 metabolism in cDNA expressed CYP enzymes. There is the possibility of drug-drug interactions when prescribing DA-8159 concomitantly with known inhibitors or inducers of CYP3A4.

3.?DA-8159 was found to be only a very weak inhibitor of eight major CYPs (1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4), the largest inhibition occurring against CYP2D6 (IC₅₀ 67.7?μM) in human liver microsomes. Drug–drug interactions would not be predicted on the basis of DA-8159 inhibiting the metabolism of coadministered drugs.     

Metabolism of deltamethrin and cis- and trans-permethrin by human expressed cytochrome P450 and carboxylesterase enzymes

1. The metabolism of the pyrethroids deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes.

2. DLM, CPM and TPM were metabolised by human CYP2B6 and CYP2C19, with the highest apparent intrinsic clearance (CL_int) values for pyrethroid metabolism being observed with CYP2C19. Other CYP enzymes contributing to the metabolism of one or more of the three pyrethroids were CYP1A2, CYP2C8, CYP2C9*1, CYP2D6*1, CYP3A4 and CYP3A5. None of the pyrethroids were metabolised by CYP2A6, CYP2E1, CYP3A7 or CYP4A11.

3. DLM, CPM and TPM were metabolised by both human CES1 and CES2 enzymes.

4. Apparent CL_int values for pyrethroid metabolism by CYP and CES enzymes were scaled to per gram of adult human liver using abundance values for microsomal CYP enzymes and for CES enzymes in liver microsomes and cytosol. TPM had the highest and CPM the lowest apparent CL_int values for total metabolism (CYP and CES enzymes) per gram of adult human liver.

5. Due to their higher abundance, all three pyrethroids were extensively metabolised by CES enzymes in adult human liver, with CYP enzymes only accounting for 2%, 10% and 1% of total metabolism for DLM, CPM and TPM, respectively.

     

Dydrogesterone metabolism in human liver by aldo-keto reductases and cytochrome P450 enzymes

1.?The metabolism of dydrogesterone was investigated in human liver cytosol (HLC) and human liver microsomes (HLM). Enzymes involved in dydrogesterone metabolism were identified and their relative contributions were estimated.

2.?Dydrogesterone clearance was clearly higher in HLC compared to HLM. The major active metabolite 20α-dihydrodydrogesterone (20α-DHD) was only produced in HLC.

3.?The formation of 20α-DHD by cytosolic aldo-keto reductase 1C (AKR1C) was confirmed with isoenzyme-specific AKR inhibitors.

4.?Using recombinantly expressed human cytochrome P450 (CYP) isoenzymes, dydrogesterone was shown to be metabolically transformed by CYP3A4 and CYP2C19.

5.?A clear contribution of CYP3A4 to microsomal metabolism of dydrogesterone was demonstrated with HLM and isoenzyme-specific CYP inhibitors, and confirmed by a significant correlation between dydrogesterone clearance and CYP3A4 activity.

6.?Contribution of CYP2C19 was shown to be clearly less than CYP3A4 and restricted to a small group of human individuals with very high CYP2C19 activity. Therefore, it is expected that CYP2C19 genetic variations will not affect dydrogesterone pharmacokinetics in man.

7.?In conclusion, dydrogesterone metabolism in the liver is dominated primarily by cytosolic enzymes (particularly AKR1C) and secondarily by CYP3A4, with the former exclusively responsible for 20α-DHD formation.     

Raloxifene glucuronidation in liver and intestinal microsomes of humans and monkeys: contribution of UGT1A1, UGT1A8 and UGT1A9

1.?Raloxifene is an antiestrogen that has been marketed for the treatment of osteoporosis, and is metabolized into 6- and 4′-glucuronides by UDP-glucuronosyltransferase (UGT) enzymes. In this study, the in vitro glucuronidation of raloxifene in humans and monkeys was examined using liver and intestinal microsomes and recombinant UGT enzymes (UGT1A1, UGT1A8 and UGT1A9).

2.?Although the K_m and CL_int values for the 6-glucuronidation of liver and intestinal microsomes were similar between humans and monkeys, and species differences in V_max values (liver microsomes, humans?>?monkeys; intestinal microsomes, humans?<?monkeys) were observed, no significant differences were noted in the K_m or S₅₀, V_max and CL_int or CL_max values for the 4′-glucuronidation of liver and intestinal microsomes between humans and monkeys.

3.?The activities of 6-glucuronidation in recombinant UGT enzymes were UGT1A1?>?UGT1A8?>UGT1A9 for humans, and UGT1A8?>?UGT1A1?>?UGT1A9 for monkeys. The activities of 4′-glucuronidation were UGT1A8?>?UGT1A1?>?UGT1A9 in humans and monkeys.

4.?These results demonstrated that the profiles for the hepatic and intestinal glucuronidation of raloxifene by microsomes were moderately different between humans and monkeys.     

Inhibitory effects of curculigoside on human liver cytochrome P450 enzymes

1.?Curculigoside possesses numerous pharmacological activities, and however, little data available for the effects of curculigoside on the activity of human liver cytochrome P450 (CYP) enzymes.

2.?This study investigates the inhibitory effects of curculigoside on the main human liver CYP isoforms. In this study, the inhibitory effects of curculigoside on the eight human liver CYP isoforms 1A2, 2A6, 2E1, 2D6, 2C9, 2C19, 2C8, and 3A4 were investigated in human liver microsomes.

3.?The results indicated that curculigoside could inhibit the activity of CYP1A2, CYP2C8, and CYP3A4, with IC₅₀ values of 15.26, 11.93, and 9.47?μM, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that curculigoside was not only a noncompetitive inhibitor of CYP1A2, but also a competitive inhibitor of CYP2C8 and CYP3A4, with Ki values of 5.43, 3.54, and 3.35?μM, respectively. In addition, curculigoside is a time-dependent inhibitor for CYP1A2, with kinact/K_I values of 0.056/6.15?μM^?1?min^?1.

4.?The in vitro studies of curculigoside with CYP isoforms suggest that curculigoside has the potential to cause pharmacokinetic drug interactions with other coadministered drugs metabolized by CYP1A2, CYP2C8, and CYP3A4. Further in vivo studies are needed in order to evaluate the significance of this interaction.     

Comparative study of the metabolism of drug substrates by human cytochrome P450 3A4 expressed in bacterial,yeast and human lymphoblastoid cells

1. The aim was to compare the metabolic activity of human CYP3A4 expressed in bacteria (E. coli), yeast (S. cerevisiae) and human lymphoblastoid cells (hBl), with the native CYP3A4 activity observed in a panel of human livers. 2. Three CYP3A4 substrates were selected for study: dextromethorphan (DEM), midazolam (MDZ) and diazepam (DZ). The substrate metabolism in each of the four systems was characterized by deriving the kinetic parameters K_m or S₅₀, V_max and intrinsic clearance (CL_int) or maximum clearance (CL_max) from the kinetic profiles; the latter differing by 100-fold across the three substrates. 3. The K_m or S₅₀ for the formation of metabolites 3-methoxymorphinan (MEM), 1'-hydroxymidazolam (1'-OH MDZ) and 3-hydroxydiazepam (3HDZ) compared well in all systems. For CYP3A4-mediated metabolism of DEM, MDZ and DZ, the V_max for hBl microsomes were generally 2-9-fold higher than the respective yeast and human liver microsomes and E. coli membrane preparations, resulting in greater CL_int or CL_max. In the case of 3HDZ formation, non-linear kinetics were observed for E. coli, hBl microsomes and human liver microsomes, whereas the kinetics observed for S. cerevisiae were linear. 4. The use of native human liver microsomes for drug metabolic studies will always be preferable. However, owing to the limited availability of human tissues, we find it is reasonable to use any of the recombinant systems described herein, since all three recombinant systems gave good predictions of the native human liver enzyme activities.     

Identification of human cytochrome P(450)s that metabolise anti-parasitic drugs and predictions of in vivo drug hepatic clearance from in vitro data

Objective Knowledge about the metabolism of anti-parasitic drugs (APDs) will be helpful in ongoing efforts to optimise dosage recommendations in clinical practise. This study was performed to further identify the cytochrome P₄₅₀ (CYP) enzymes that metabolise major APDs and evaluate the possibility of predicting in vivo drug clearances from in vitro data.Methods In vitro systems, rat and human liver microsomes (RLM, HLM) and recombinant cytochrome P₄₅₀ (rCYP), were used to determine the intrinsic clearance (CL_int) and identify responsible CYPs and their relative contribution in the metabolism of 15 commonly used APDs.Results and discussion CL_int determined in RLM and HLM showed low (r²=0.50) but significant (P<0.01) correlation. The CL_int values were scaled to predict in vivo hepatic clearance (CL_H) using the 'venous equilibrium model'. The number of compounds with in vivo human CL data after intravenous administration was low (n=8), and the range of CL values covered by these compounds was not appropriate for a reasonable quantitative in vitro–in vivo correlation analysis. Using the CL_H predicted from the in vitro data, the compounds could be classified into three different categories: high-clearance drugs (>70% liver blood flow; amodiaquine, praziquantel, albendazole, thiabendazole), low-clearance drugs (<30% liver blood flow; chloroquine, dapsone, diethylcarbamazine, pentamidine, primaquine, pyrantel, pyrimethamine, tinidazole) and intermediate clearance drugs (artemisinin, artesunate, quinine). With the exception of artemisinin, which is a high clearance drug in vivo, all other compounds were classified using in vitro data in agreement with in vivo observations. We identified hepatic CYP enzymes responsible for metabolism of some compounds (praziquantel—1A2, 2C19, 3A4; primaquine—1A2, 3A4; chloroquine—2C8, 2D6, 3A4; artesunate—2A6; pyrantel—2D6). For the other compounds, we confirmed the role of previously reported CYPs for their metabolism and identified other CYPs involved which had not been reported before.Conclusion Our results show that it is possible to make in vitro–in vivo predictions of high, intermediate and low CL_int drug categories. The identified CYPs for some of the drugs provide a basis for how these drugs are expected to behave pharmacokinetically and help in predicting drug–drug interactions in vivo.     

Approach to the prediction of the contribution of major cytochrome P450 enzymes to drug metabolism in the early drug-discovery stage

It is important to determine the cytochrome P450 (CYP) contribution of certain drugs by taking into consideration the attrition due to issues such as genetic polymorphism and inter-individual variation. In many cases in the early discovery stage, the metabolites of a new chemical have not been identified. Therefore, the present paper devised an approach in which the in vitro intrinsic clearance (CL_int) value for new chemicals was determined by measuring substrate depletion. The following prediction methods were compared to calculate CL_int using data from recombinant CYP enzymes: (1) the relative CYP content in human liver microsomes; (2) the relative activity factor (RAF) based on the V_max value; and (3) the RAF value based on the CL_int value. The most accurate prediction method was RAF based on CL_int. This method would be useful in the early drug-discovery process in cases in which the main metabolite is not identified.     

Effect of buffer conditions on CYP2C8-mediated paclitaxel 6α-hydroxylation and CYP3A4-mediated triazolam α- and 4-hydroxylation by human liver microsomes

Abstract

1.?Buffer conditions in in vitro metabolism studies using human liver microsomes (HLM) have been reported to affect the metabolic activities of several cytochrome P450 (CYP) isozymes in different ways, although there are no reports about the dependence of CYP2C8 activity on buffer conditions.

2.?The present study investigated the effect of buffer components (phosphate or Tris-HCl) and their concentration (10–200?mM) on the CYP2C8 and CYP3A4 activities of HLM, using paclitaxel and triazolam, respectively, as marker substrates.

3.?The K_m (or S₅₀) and V_max values for both paclitaxel 6α-hydroxylation and triazolam α- and 4-hydroxylation, estimated by fitting analyses based on the Michaelis–Menten or Hill equation, greatly depended on the buffer components and their concentration.

4.?The CL_int values in phosphate buffer were 1.2–3.0-fold (paclitaxel) or 3.1–6.4-fold (triazolam) higher than in Tris-HCl buffer at 50–100?mM. These values also depended on the buffer concentration, with a maximum 2.3-fold difference observed between 50 and 100?mM which are both commonly used in drug metabolism studies.

5.?These findings suggest the necessity for optimization of the buffer conditions in the quantitative evaluation of metabolic clearances, such as in vitro–in vivo extrapolation and also estimating the contribution of a particular enzyme in drug metabolism.     

Preclinical evaluation of the potential for cytochrome P450 inhibition and induction of the selective ALK inhibitor,alectinib

1.?A novel selective anaplastic lymphoma kinase (ALK) inhibitor, alectinib, has shown remarkable efficacy and safety in patients with ALK-positive non-small-cell lung cancer (NSCLC). The purpose of this study was to evaluate in vitro the potential to inhibit and induce cytochrome P450 (CYP) isoforms for alectinib and its major metabolite M4.

2.?Alectinib and M4 did not show the meaningful direct inhibition of six major CYP isoforms (CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A4) in human liver microsomes (HLM). Alectinib, but not M4, competitively inhibited CYP2C8, by which few marketed drugs are exclusively metabolized, with an inhibition constant of 1.98?μM.

3.?Out of the seven CYP isoforms in HLM, alectinib and M4 showed time-dependent inhibition (TDI) of only CYP3A4, which suggests low TDI potential due to low inactivation efficiency.

4.?Alectinib exhibited quite smaller induction of mRNA expression of CYP1A2, 2B6 and 3A4 genes in human hepatocytes compared to the respective positive controls, suggesting a low potential of enzyme induction.

5.?In summary, the risk of alectinib causing drug-drug interactions with coadministered drugs is expected to be low due to the weak potential of CYP inhibition and induction estimated in the preclinical studies.     

Enantioselective N-Demethylation and Hydroxylation of Sibutramine in Human Liver Microsomes and Recombinant Cytochrome P-450 Isoforms

The enantioselective metabolism of sibutramine was examined using human liver microsomes (HLM) and recombinant cytochrome P-450 (CYP) isoforms. This drug is metabolized to N-mono-desmethyl- (M1) and N,N-di-desmethylsibutramine (M2), and subsequent hydroxylation results in hydroxyl M1 (HM1) and hydroxyl M2 (HM2). No significant difference was noted in formation of M1from sibutramine between R- and S-sibutramine in HLM. However, S-enantiomers of M1 and M2 were preferentially metabolized to M2, HM1, and HM2compared to R-enantiomers in HLM, and intrinsic clearance (Cl_int) ratios of S-enantiomers/R-enantiomers were 1.97, 4.83, and 9.94 for M2, HM1, and HM2, respectively. CYP3A4 and CYP3A5 were only involved in the formation of M1, whereas CYP2B6 and CYP2C19 were responsible for all metabolic reactions of sibutramine. CYP2C19 and CYP3A5 displayed catalytic preference for S-sibutramine to S-M1, whereas CYP2B6 and CYP3A4 showed little or no stereoselectivity in metabolism of sibutramine to M1. In the case of M2 formation, CYP2B6 metabolized S-M1 more rapidly than R-M1 with a Cl_int ratio of 2.14. However, CYP2C19 catalyzed less S-M1 than R-M1 and the Cl_int ratio of S-M1 to R-M1 was 0.65. The most significant enantioselectivity was observed in formation of HM1 from M1, and HM2 from M2. CYP2B6 and CYP2C19 exhibited preferential catalysis of formation of hydroxyl metabolites from S-enantiomers rather than R-enantiomers. These results indicate that S-sibutramine was more rapidly metabolized by CYP isoforms than R-sibutramine, and that enantioselective metabolism needs to be considered in drug interactions involving sibutramine and co-administered drugs.     

Contribution of cytochrome P450 3A4 and 3A5 to the metabolism of atorvastatin

Atorvastatin is a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor that is mainly metabolized by cytochrome P450 (CYP) 3A4. A recent study showed that the lipid-lowering effect of statins is affected by the CYP3A5 polymorphism. Therefore, it was investigated whether CYP3A5 contributes to the metabolism of atorvastatin. Two metabolites of atorvastatin, para- and ortho-hydroxyatorvastatin, were produced by human liver microsomes and human recombinant CYP3A enzymes, and the enzyme kinetic pattern exhibited substrate inhibition. The intrinsic clearance (CL_int) rates of para- and ortho-hydroxyatorvastatin by CYP3A4 were 2.4- and 5.0-fold of the respective CL_int rates of CYP3A5, indicating that CYP3A4 is the major P450 isoform responsible for atorvastatin metabolism. These results suggest that atorvastatin is preferentially metabolized by CYP3A4 rather than by CYP3A5, and thus the genetic CYP3A5 polymorphism might not be an important factor in the inter-individual variation of atorvastatin disposition and pharmacodynamics in human.     

Prediction of metabolic clearance of diclofenac in adjuvant-induced arthritis rats using a substrate depletion assay

1.?The purpose of this study was to evaluate drug clearance measured by the metabolic intrinsic clearance (CL_int) in a substrate depletion assay in comparison with the in vivo clearance (CL_tot) observed in adjuvant-induced arthritis (AA) rats. 2.?After intravenous administration of diclofenac as a model drug, CL_tot was 2.8-fold higher in AA rats than in control rats. In two different substrate depletion assays with liver microsomes for glucuronidation and hydroxylation, the CL_int values for glucuronidation was significantly decreased in AA rats to 60% of the value in control rats, whereas the CL_int values for hydroxylation were similar. The unbound fraction of diclofenac in plasma (f_{u, plasma}) was significantly higher (2.8-fold) in AA rats than in control rats. 3.?Hepatic clearance predicted from the CL_int values for both biotransformation pathways and f_{u, plasma} was higher in AA rats than in control rats, with good consistency between predicted and observed values. The same results were obtained for experiments using hepatocytes. 4.?The plasma protein-binding activities, rather than metabolic clearance, in both types of rats would be a determining factor in the pharmacokinetic behaviour differences between control and AA rats. 5.?In summary, substrate depletion assays with liver microsomes and hepatocytes in combination with protein binding assessment can help to predict changes in pharmacokinetics under AA conditions.